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Evaluating Seed Viability by an Unimbibed Seed Crush Test in Comparison with theTetrazolium TestAuthor(s): Jeremiah T. Sawma and Charles L. MohlerSource: Weed Technology, Vol. 16, No. 4 (Oct. - Dec., 2002), pp. 781-786Published by: Weed Science Society of America and Allen PressStable URL: http://www.jstor.org/stable/3989152 .Accessed: 04/03/2011 10:48

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Weed Technology. 2002. Volume 16:781-786

Evaluating Seed Viability by an Unimbibed Seed Crush Test in Comparison with the Tetrazolium Test1

JEREMIAH T. SAWMA and CHARLES L. MOHLER2

Abstract: The unimbibed crush test, in which seed viability is evaluated by crushing and visual inspection of dry seeds, was compared with tetrazolium staining, an established method of testing seed viability. The unimbibed crush test potentially provides an immediate and rapid method for determining seed viability. Six sets of seed lots, involving the four weed species, velvetleaf, common lambsquarters, redroot pigweed, and smooth pigweed, were tested by each method. For four of the six sets, results from the crush test were statistically indistinguishable from those of the tetrazolium test. For the other two sets of seed lots, the crush test indicated higher viability than did the tetrazolium test. The crush test may be most useful for seed bank surveys in which many samples are typically processed, and most of the variation in density of viable seeds is associated with number of seeds present rather than percentage viability. Its use in more exacting circumstances like seed survival studies requires caution. Nomenclature: Tetrazolium, 2,3,5 triphenyl tetrazolium chloride; common lambsquarters, Cheno- podium album L. #3 CHEAL; redroot pigweed, Amaranthus retroflexus L. # AMARE; smooth pigweed, Amaranthus hybridus L. # AMACH; velvetleaf, Abutilon theophrasti Med. # ABUTH.

INTRODUCTION

Seed viability testing is important for understanding and managing plants that have persistent seed banks. Be- cause these species produce many seeds that survive for several years in the soil, the majority of individuals in such a population exist as seeds. Common lambsquarters, one such species, was found at a density of 5,930 seeds/M2 after 5 yr without reproduction at the site (Har- ris 1959). In another study, all weed seed input to several fields was prevented by the use of herbicides for 5 yr. After weed control was relaxed, numerous grass and broadleaf weed species established from the seed bank (Bumside et al. 1986). Because live seeds are sometimes visually indistinguishable from dead seeds, testing for seed viability is required to estimate the number of living seeds in a seed bank.

A viable seed is a seed that has the potential to germinate. Viability is the percentage or proportion of viable seeds in a seed lot. Several methods have been used

' Received for publication January 18, 2002, and in revised form June 17, 2002.

2 Research Assistant and Senior Research Associate, Department of Ecol- ogy and Evolutionary Biology, Corson Hall, Cornell University, Ithaca, NY 14853. Current address of J. T. Sawma: Appalachian lab, 901 Braddock Road, Frostburg, MD 21532; current address of C. L. Mohler: Department of Crop and Soil Sciences, 907 Bradfield Hall, Cornell University, Ithaca, NY 14853. Corresponding author's E-mail: clml @cornell.edu.

3 Letters following this symbol are a WSSA-approved computer code from Composite List of Weeds, Revised 1989. Available only on computer disk from WSSA, 810 East 10th Street, Lawrence, KS 66044-8897.

to estimate viability, but all are laborious enough to limit the scope of seed bank studies. Viability testing has been used in a wide variety of studies, including many on agricultural weeds (Forcella and Lindstrom 1988; Froud- Williams et al. 1983, 1984; Goss 1985; Harradine 1986; Leguizamon 1986; Leuschen and Anderson 1980; Lewis 1958; Moss 1985; Roberts and Dawkins 1967; Roberts and Feast 1973), restoration and conservation ecology (Bertiller and Aloia 1997; Dalling and Denslow 1998; Fischer and Matthies 1998), and natural ecosystems, particularly those with frequent fires (Ashton et al. 1998; Morgan 1998; Whittle et al. 1998).

In these articles, seed viability testing was accom-, plished by counting the seedlings emerging from soil- filled flats (Forcella and Lindstrom 1988; Froud-Wil- liams et al. 1983, 1984; Goss 1985; Harradine 1986; Le- guizamon 1986; Leuschen and Anderson 1980; Lewis 1958; Moss 1985), by testing the seeds with tetrazolium, which stains respiring tissues red (Gross 1990; Harradine 1986; Leguizam6n 1986), or by examination of imbibed seeds for signs of decay (Froud-Williams et al. 1984; Leuschen and Anderson 1980; Lewis 1958). Viability testing with tetrazolium or by examination for signs of decay is always preceded by a brief germination period to decrease the number of seeds that must be evaluated with these methods.

Each test has advantages and disadvantages. Germi-

781

SAWMA AND MOHLER: EVALUATING SEED VIABILITY

nation of seeds from soil in flats does not require sepa- ration of seeds from the soil, but it may require up to 2 yr to induce germination of all seeds (Roberts and Dawkins 1967; Roberts and Feast 1973). Typically, conditions are manipulated to encourage germination, but some seeds may still remain dormant, and some seeds may die during the study. Tetrazolium testing provides a quicker way to obtain seed viability data, but it is laborious, and its application to small seeds requires much skill. The International Seed Testing Association (1985) details the criteria for a crush test on imbibed seeds, in which the seeds are crushed and examined for decay after germination of nondormant individuals. This test is faster than the tetrazolium test, but it is still time-con- suming because germinable seeds must be sprouted and removed first.

Several studies have used a crush test on unimbibed seeds. In these studies, seeds were crushed, and those that were brittle or discolored were classified as nonviable (Warnes and Anderson 1984; Wilson 1985; Wilson and Lawson 1992). This article compares the unimbibed crush test with the tetrazolium test to assess the reliabil- ity of the crush test as a method for determining seed viability. The unimbibed crush test is the quickest of all the tests and notably faster than the imbibed crush test, which usually involves initial removal of germinable individuals. For comparison, tetrazolium staining was cho- sen because it is generally considered to be the most accurate method for determining seed viability. Com- parisons with other methods were not included in the study because of the additional labor required and the insufficient number of seeds in many of the seed lots used in the study.

MATERIALS AND METHODS

The tetrazolium and crush tests were compared in six sets of seed lots. Each seed lot represented a seed population with a separate history. Seed lot sets differed with respect to weed species and the means that were used to create variation in viability among seed lots. In all, a total of 171 seed lots were evaluated, and these included four agricultural weed species: velvetleaf, common lambsquarters, redroot pigweed, and smooth pigweed.

Sources of Seed Lots. All seed lots underwent treat- ments expected to reduce their initial viability and produce a range of viabilities among seed lots. A wide range of viabilities was desirable because the crush test might approximate the tetrazolium test only within a certain

range of viability. Sets of seed lots of velvetleaf, common lambsquarters, and redroot pigweed were taken from a seed survival study (Mohler 1999), in which seeds were buried at different depths and left in the soil for 6 mo. At the end of the 6-mo period, seeds were recovered by elutriation (Gross and Renner 1989), dried at 40 C, and refrigerated at 4 C until testing. Each seed lot corresponded to the seeds recovered from a single microplot in that experiment. Because these seed lots were exposed to a range of conditions that were expected to affect seed mortality and germination, it was expected that individual seed lots would vary widely in viability. But most of the nonviable seeds in these seed lots had decayed to the point that they were determined to be nonviable by visual inspection, making both the tetrazolium test and the crush test unnecessary for these seeds. The remaining seeds in each seed lot were tested by both methods. Nearly all were viable according to the tetrazolium test.

These sets of seed lots of velvetleaf, common lambsquarters, and redroot pigweed derived from the buried seed study (Mohler 1999) are henceforth referred to as the Narrow Range of Viability sets. Although these sets of seed lots did not provide the desired range of viabilities, they were still useful for comparing the two tests in the common situation where seed viability is high.

To provide data with a broad range of viability, including some low-viability seed lots, three more sets of seed lots were generated in the laboratory. Each of the seed sources used to generate these seed lots consisted of either seeds from a single source population collected at one time or purchased seeds from a commercial source. Seed lots were generated by germinating seeds from each source both in soil and in distilled water in petri dishes. Thus, for example, common lambsquarters seeds collected near Ithaca, NY, in 1997 produced two seed lots: one from residual seeds after petri dish germination and one from residual seeds after soil germination. All seedlings were removed as they sprouted. At the end of the germination period, seeds were recovered and dried at 40 C. The seed lots produced in this way differed in viability because the remaining nongerminat- ed seeds varied in the proportion that were viable but dormant vs. the proportion that were not viable. Some of this variability was inherent in the original bulk seed collections, and some occurred because a greater proportion of seeds tended to germinate in soil than in distilled water, thereby producing different ratios of viable dormant to nonviable seeds. These sets of seed lots of velvetleaf, pigweed, and common lambsquarters were called the Broad Range of Viability sets.

782 Volume 16, Issue 4 (October-December) 2002

WEED TECHNOLOGY

Broad Range of Viability pigweed included seed lots of both redroot pigweed and smooth pigweed because only a few seed lots from each species were available. Combining data from the two species was reasonable because they are closely related, and the relative perfor- mance of the two viability tests did not differ (see data analysis below).

Viability Testing. All seeds were first visually assessed beneath a dissecting microscope, and the badly deteriorated seeds were removed. These included seeds that were very moldy or empty (as viewed through a hole in the seed coat) or showed seed coat damage associated with germination. The appearance of seeds that died during germination was determined experimentally by inspection of seeds that were desiccated at various stages of germination. In pigweed and velvetleaf, germination damaged the seed coat where the radicle emerged and sometimes caused the seed coat to fold in on itself. In common lambsquarters, germination damage was appar- ent when the two halves of the seed coat were split apart slightly. Seeds showing germination damage were all empty, but this was usually discovered only after the germination damage was noticed.

After removing the deteriorated seeds, subsamples of 40 seeds from each seed lot were tested with the crush test, and another 40 were tested with tetrazolium. For a few seed lots with less than 80 seeds, each test was performed on half the available seeds for the Broad Range of Viability seed lots. For the Narrow Range of Viability seed lots, 40 seeds were tested with tetrazolium and the remaining seeds with the crush test. One Narrow Range of Viability velvetleaf seed lot was eliminated because only four seeds were available for crush testing, although including that lot would not have changed the mean viability or conclusions. Of the 171 seed lots included in the study, over 35 seeds were tested by both methods for 157 lots. Only three lots had fewer than 15 seeds for one or the other test. The minimum involved one of the Broad Range of Viability velvetleaf seed lots for which eight seeds were tested by tetrazolium and nine seeds were crush tested.

The crush test was performed according to the following procedures. A single seed was folded in a small sheet of paper and struck once with a wrench with sufficient force to break but not pulverize the seed. Velvetleaf seeds were classified as viable if they showed creamy and oily flesh and nonviable if they crumbled and appeared brown or black. Pigweed and common lambsquarters seeds were classified as viable if their flesh appeared creamy and oily and as nonviable if they ap-

peared powdery, black or brown when crushed. When the Broad Range of Viability data sets were tested, it was discovered that all pigweed and common lambsquarters seeds that were powdery, black or brown could be crushed beneath the flat side of a pair of forceps when firm pressure was applied. This method of crushing was then adopted for the Broad Range of Viability pigweed and common lambsquarters seeds. The two methods produced equivalent data, but the latter required less con- centration to obtain a consistent degree of seed breakage.

The tetrazolium test was performed according to standard procedures (Intermational Seed Testing Association 1985). Seeds were placed in petri dishes on double sheets of Watman No. 1 filter paper moistened with distilled water and left on a laboratory bench for 4 d. Vel- vetleaf seeds were first pricked with a needle to break any hard seed coats and then covered with an additional sheet of moistened filter paper to ensure that the seeds imbibed. After 4 d the germinants were removed and counted. The remaining seeds were sectioned to expose the embryo and stained with 1% solution by weight of tetrazolium. After 5 h, seeds whose embryos had stained red and had firm flesh were classified as viable. Propor- tion viable was computed as the sum of the number germinating plus the number staining positive divided by the total number tested.

Data Analysis. Proportion of viable seeds was transformed using y = arcsine(V/x) to improve normality. To test for bias in the crush test, values from the two procedures were compared using a Wilcoxon rank test for the Narrow Range sets of seed lots and a paired t test for the Broad Range sets of seed lots. To determine whether the crush test produced more variable results than did the tetrazolium test, variances for the two viability tests were compared (Snedecor and Cochran 1967).

Regressions of proportion viable by the tetrazolium test vs. proportion viable by the crush test were con- ducted for the Broad Range of Viability seed lots. The slopes and intercepts of the regression lines and the variance of residuals of redroot pigweed and smooth pigweed were compared, and because none of these differed significantly, the two sets of seed lots were combined. Slopes of the regression for velvetleaf, common lambsquarters, and the combined pigweeds were then calculated with the intercept fixed at the origin. Variation in data tends to reduce the slope of a regression line when the intercept is estimated. Therefore, the lines were fitted through the origin to allow comparison with a line of slope 1, the expected slope if the two viability tests pro-

Volume 16, Issue 4 (October-December) 2002 783


Table 1. Comparison of tetrazolium and crush tests for estimating seed viability of weed seeds. Means were calculated from untransformed proportions. Means and standard deviations of arcsine (\x) transformed values are shown in parentheses.

Type of set Mean proportion of seeds viable

Species of seed lots Na Tetrazolium test Crush test Significanceb

Redroot pigweed Narrow range 19 0.77 (1.10 ? 0.19) 0.77 (1.10 ? 0.18) NS Pigweedc Broad range 15 0.63 (0.95 ? 0.31) 0.58 (0.89 ? 0.36) NS Common lambsquarters Narrow range 53 0.90 (1.31 ? 0.19) 0.94 (1.39 ? 0.17) ** Common lambsquarters Broad range 20 0.46 (0.75 ? 0.41) 0.49 (0.77 + 0.41) NS Velvetleaf Narrow range 55 1.00 (1.56 ? 0.04) 0.99 (1.55 ? 0.08) NS Velvetleaf Broad range 9 0.51 (0.80 ? 0.23) 0.75 (1.08 ? 0.22) *

a Number of seed lots tested. bSignificance of difference in mean viability of transformed values: **, P < 0.01; *, P < 0.05; NS, not significant. c Both redroot pigweed seed lots and smooth pigweed seed lots were included.

1 - a. Pigweed spp.

0.8-

0.6-

0.4-

o Redroot

* Smooth

u, 0

1- b. Common - 08 lambsquarters . 0.8 0 c 0 'e 0.6- 0 1

0.4-

0 0 0.4

1 c. Velvetleaf

0.8(

0.6-

0.4-

0 0.2 0.4 0.6 0.8

Crush test (proportion of viable seeds)

Figure 1. Proportion of viable seeds as evaluated by the tetrazolium test vs. proportion of viable seeds as evaluated by the crush test for the Broad Range of Viability sets of seed lots of (a) pigweed species, (b) common lambsquarters, and (c) velvetleaf. The expected relationship if the tests were equivalent (i.e., y =x) appears as a straight line on each graph.

duced identical results. Slopes were tested against the value 1 using a t test (Snedecor and Cochran 1967).

RESULTS AND DISCUSSION

Mean proportion of seeds estimated as viable by the crush and tetrazolium tests was similar in four sets of seed lots: Narrow Range of Viability velvetleaf and pigweed, and Broad Range of Viability pigweed and common lambsquarters (Table 1). The two tests produced similar results over a wide range of viability for the Broad Range of Viability pigweed and common lambsquarters sets of seed lots (Figure 1). Regression analyses were not useful for Narrow Range of Viability pigweed, lambsquarters, and velvetleaf because of a restricted range of viabilities. The range of viability for the sets of seed lots may be judged from the standard deviations given in Table 1.

Slopes of the regression lines for all three Broad Range of Viability seed lots were tested, and none of them differed significantly from 1 at a confidence level of 0.05. This indicated that the tetrazolium test and the crush test produced identical results. Although the test of slope agrees with the graphed data for the pigweeds and common lambsquarters (Figures la and lb), the regression of velvetleaf (Figure 1c) appears to have a slope of less than 1. The slope for Broad Range of viability velvetleaf probably did not differ significantly from 1 because relatively few seed lots were available for testing, and the data show wide variation.

Variance of the crush test did not differ from variance of the tetrazolium test for any of the data sets (see standard deviations in Table 1). This demonstrated that nei- ther test has significantly better precision.

In two data sets, Narrow Range common lambsquarters and Broad Range velvetleaf, the crush test found higher viability than did the tetrazolium test (Table 1; Figure lc). Assuming that the tetrazolium test gave a


WEED TECHNOLOGY

correct estimate, this suggests that the crush test, when it errs, tends to misclassify nonviable seeds as viable. The source of the difference between the two tests for Narrow Range common lambsquarters is unclear. The difference in viability by the two tests for this set of seed lots was small and probably only detectable because of the many seed lots tested (Table 1).

Microbial attack probably caused the discrepancy in Broad Range of Viability velvetleaf. Bacteria attacked velvetleaf seeds while nondormant seeds were being removed by germination in petri dishes during initial prep- aration of Broad Range of Viability seed lots. Bacterial attack was not an issue during tetrazolium staining because the seeds were only kept moist for 4 d. Many of the attacked seeds were in the process of dying when collected and dried before testing, as subsequently indicated by weakly positive tetrazolium tests. A seed was weakly positive when the tetrazolium test showed that it had some live tissue, but it probably lacked sufficient vigor for germination. These seeds were considered nonviable. The structure of the embryos of weakly positive seeds was still intact, however, so the crush test indicated that such seeds were viable. These results suggest that the tetrazolium test would be a better choice for a seed bank that is experiencing substantial mortality when it is sampled.

Although the crush test sometimes showed a significant difference in mean viability from the tetrazolium test, it has several useful advantages. First, the crush test can be used to recognize seeds that died during germination. For pigweed and common lambsquarters, seeds that had germinated to the point of protrusion of the radicle appeared powdery when crushed. Samples that were imbibed and deliberately desiccated shortly before the radicle protrusion was expected also showed a high rate of powdery seeds. For the Broad Range of Viability pigweed and common lambsquarters, seed viability by the tetrazolium test was equivalent to that of the crush test, with powdery seeds scored as dead. Thus, powdery tissue in pigweed and common lambsquarters probably indicates (1) that the seed was dead, and (2) that death occurred after initiation of germination and most likely by desiccation.

Powdery seeds were observed primarily in the Broad Range of Viability sets of seed lots in which germination may have been terminated during the viability reduction procedure. Very few powdery seeds were found in the Narrow Range of Viability sets of seed lots, which were dug late in the season after germination for the year was complete or early in spring before germination began.

For field samples, powdery endosperm is probably a sign that germination was stopped when the seeds were collected, or that germination was initiated and then stopped during the soil collection and seed extraction procedure. The ability to recognize seeds that are germinating is useful because it indicates either that the density of seeds in the seed bank was rapidly changing when the sample was dug or that the samples were mishandled.

Second, the labor required for crush testing is typically about half that of tetrazolium testing. Moreover, only seeds that fail to germinate after some period of moistening in a petri dish are actually stained with tetrazolium, so if few seeds germinate, then a large percentage must be sectioned and stained. In such cases the crush test is comparatively even faster. Tetrazolium testing is particularly laborious for species with an imper- meable seed coat like velvetleaf because seeds must be treated first to allow imbibition and subsequent removal of germinants. In addition, sectioning hard seeds is im- practical, and unimbibed seeds will not stain properly.

Third, sectioning small seeds for the tetrazolium test without destroying them is difficult. During tetrazolium testing, seeds must be partially sectioned to allow the embryo to contact the dye.

Finally, although the tetrazolium test is apparently more accurate sometimes than the crush test, it too has potential sources of error. These include false positives due to dark red precipitates produced by microbial res- piration (International Seed Testing Association 1985) and false negatives for small-seeded species due to the embryo separating from the rest of the seed, thereby causing the seed to appear nonviable. Very careful work that includes microscopic examination of every stained seed can minimize these sources of error, but such at- tention to detail is rarely practical when testing many seed lots.

Because the unimbibed crush test is much less laborious than the tetrazolium test, it would be a useful sub- stitute for the tetrazolium test in studies where the risk of some error is acceptable. This will frequently be the case in seed bank survey work because variation in the total number of seeds is often large, whereas variation in the proportion viable is often relatively small, with most seeds being viable. For example, in a seed bank survey of agricultural fields of the Po Valley, Italy, total seed density ranged from 1,660 to 53,371 seeds/M2 (Zan- in et al. 1992). In another study, seed density ranged from 1,500 to 67,000 seeds/M2 (Roberts and Chancellor 1986). If seed densities vary by orders of magnitude, occasional large errors in the estimated proportion of vi-

Volume 16, Issue 4 (October-December) 2002 785


able seeds will barely affect the relative density of viable seeds among samples. Moreover, because nonviable seeds tend to decay under natural conditions, the percentage of nonviable seeds in natural seed banks tends to be relatively low, and this in turn makes large errors in estimating viability of samples unlikely. These con- siderations indicate that although viability testing is a common practice in seed bank analysis, its value may be marginal in the context of the whole study.

The crush test is less useful for seed survival studies because less error is acceptable. In survival studies the original input of seeds is typically identical for all experimental units. Differences in total seeds and seed viability after some period of time depend entirely on mortality and germination. Because variation in the number of seeds between seed lots is small, an error of 10% could significantly affect the results of the study. For the Broad Range of Viability sets of seed lots, the sample standard deviations from the regressions were 0.15, 0.16, and 0.16 for common lambsquarters, pigweed, and velvetleaf, respectively. Therefore, two seed lots with identical viability by the tetrazolium test could easily differ by 20% because of variation in the crush test. Thus, even when the crush test shows no bias relative to the tetrazolium test (e.g., pigweeds in Table 1; Figure 1), the crush test should not be used for seed survival studies unless the degree of replication is unusually high.

ACKNOWLEDGMENTS

We thank C. E. McCulloch for advice on statistical analysis, S. Gardescu, P. Marks, M. Geber, J. Berkery, L. Yang, R. Rotjan, and anonymous reviewers for com- ments on early versions of the paper, and M. Niell, M. Glos, S. Yang, S. P. Yang, M. Franke, M. Plowe, S. Bourguignon, S. Cady, T Picard, M. Guevara, J. Powell, G. Kim, A. Shmidt, M. Laer, L. Chang, C. Ward, H. Torres-Pratts, B. Demonarco, M. O'Donnell, M. Kuhn, and I. Hong, who helped in obtaining the seed lots. This work was supported in part by funds from U.S.D.A. Na- tional Research Initiative Competitive Grants Program (Agreement 95-37315-2018) and by Hatch funds (Re- gional Project NE-92, NY(C)-183458) from the Cornell Agricultural Experiment Station.

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